This invention is generally in the area of immunodeficient mutant animals and methods of use thereof.
Assembly of immunoglobulin (Ig) heavy and light chain genes and of .alpha. and .beta. chain genes of the T cell receptor (TCR) is mediated to a large extent in developing lymphocytes by somatic recombination, in which widely separated gene segments are joined to form a complete variable region, a process known as V(D)J recombination, described by Tonegawa, S. Nature 302, 575-581 (1983). The genes for the antigen receptors are produced exclusively in lymphocytes through this recombinational process, which joins variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination occurs at seven different loci: the immunoglobulin (Ig) heavy chain, kappa and lambda light chain loci in B lymphocytes (Tonegawa, 1983) and the T cell receptor (TCR) alpha, beta, gamma and delta chain loci in T lymphocytes (Davis, M. M. and Bjorkman, P. J. Nature 334, 395-402 (1988)).
The prevailing model is that a common recombinase is active in precursors of both B and T cells (Yancopoulos, G. D. et al., Cell, 44:251-259 (1986)), and that the sequential recombinations are executed by developmentally controlled targeting of the recombinase to the different loci (Alt, F. W., et al., Immunol. Rev. 89, 5-30 (1986)). The assembly process is tightly regulated, occurring in a preferred temporal order (D to J joins occur before V to D, lambda segments rearrange before kappa and in a lineage specific manner (loci recombined in T cells are never fully rearranged in B cells). Developing B cells and T cells rearrange distinct gene segment families in a well-defined temporal order. In developing B cells, the heavy chain locus is rearranged before the light chain loci. Maki, R. et al., Science, 209:1366-1369 (1980); Perry, R. P. et al., Proc. Natl. Acad. Sci., USA, 78:247-251 (1981); Alt, F. et al., Cell, 27:381-390 (1981); Alt, F. et al., EMBO J., 3:1209-1219 (1984); Reth, M. G. et al., Nature, 317:353-355 (1985). In developing T cells, the .beta. chain locus is rearranged before the .alpha. chain locus. Raulet, D. H. et al., Nature, 314:103-107 (1985); Snodgrass, H. R. et al., Nature, 315:232-233 (1985); Samelson, L. E. et al., Nature, 315:765-768 (1985).
The complex mechanisms regulating V(D)J recombination are not well understood. Rearrangements are mediated by recombination signal sequences (RSSs) that flank all recombinationally competent, V, D and J gene segments. These signals are conserved among the different loci and species that carry out V(D)J recombination and are functionally interchangeable. RSSs, necessary and sufficient to direct recombination, consist of a syad-symmetric heptamer, an AT-rich nonamer and an intervening spacer region of either 12 or 23 bp. The two spacer lengths define two different RSSs and one of each is required for efficient joining to occur.
A number of activities must be involved in the joining reaction; these include the recognition of RSSs, enconucleolytic cleavage at or near the signal border, base trimming and addition (the joints of coding sequences are imprecise) and ligation of the cleaved ends (Sakano, H. et al. Nature, 280:288-294 (1979); Max, E. E. et al., Proc. Natl. Acad, Sci., USA, 76:3450-3454 (1979); Early, P. et al., Cell, 19:981-992 (1980); Sakano, H. et al., Nature, 290:562-565 (1981); Davis, M. M., Annu. Rev. Immunol., 3:537-560 (1985). Gene segments flanked by joining signals with 12 bp spacers are joined only to gene segments flanked by joining signals with 23 bp spacers. Although different sets of genes are rearranged in developing B and T cells, exogenously introduced T cell receptor gene segments can be efficiently recombined in pre-B cells. This suggests that B and T cell lineages use the same recombination machinery, as discussed by Yancopoulos, G. D. et al., (1986).
The cis-acting sequences required for V(D)J recombination have been described in detail (Tonegawa, 1983; Hesse et al., Genes Dev. 3, 1053-1061 (1989). A gene called the recombination activation gene RAG-1 has been isolated by virtue of its ability to activate V(D)J recombination in NIH 3T3 fibroblasts on an artificial recombination substrate carrying selectable markers (Schatz and Baltimore, 1988; Schatz, D. G., et al. Cell 59, 1035-1048 (1989)). A second, structurally unrelated gene called RAG-2 was later identified in the immediate vicinity of RAG-1 (Oettinger, et al., Science 248, 1517-1523 (1990).
A model has been proposed by Oettinger et al., (1990), in which RAG-1 and RAG-2 together are sufficient to induce V(D)J recombination in fibroblasts. The expression of both genes is concordant and restricted to cell lines displaying V(D)J recombination activity and developing lymphoid tissues (Schatz et al., 1989; Oettinger et al., 1990; Boehm, T. and Rabbitts, T. Proc. Natl. Acad. Sci. USA (1991); Turka et al., Science 253, 778-781 (1991). The only reported discordancies in their expression patterns are transcription of only RAG-1 in the central nervous system of the mouse, reported by Chun, et al., Cell 64, 189-200 (1991), and of only RAG-2 in the bursa of Fabricius of the chicken, reported by Carlson, et al. Cell 64, 201-208 (1991).
An animal model first described by Bosma et al., Nature 301, 527-530 (1983), for severe combined immunodeficiency (the scid mouse) has been reported that has been utilized for a number of different studies, including expression of human antibodies following injection of human lymphocytes into the mice, as described for example by McCune, J. M. Curr. Op. Immunol. 3, 2, 224-228 (1991). As reported by Bosma, G. C., et al., J. ExP. Med. 167, 1016-1033 (1988), the scid mouse, however, does produce some mouse immunoglobulin and some T cells, due to mostly aberrant rearrangements. Flow cytometric analysis of lymphoid organs reveals a blockade of lymphocyte differentiation at an immature stage, as further reviewed by Bosma, M. J. and Carroll, A. M. Annu. Rev. Immunol. 9, 323-350 (1991).
It is therefore an object of the present invention to produce an animal that is totally unable to produce functional immunoglobulin or T cell receptors.
It is a further object of the present invention to determine whether both RAG-1 and RAG-2 are required in vivo for expression of functional immunoglobulin and T cell receptors.